PREFI LEC: GENE MUTATIONS

Question 1

Permanent alteration in the DNA sequence that makes up a gene

Answer 1

GENE MUTATION

Question 2

Causes of GENE MUTATION?

Answer 2

errors in DNA replication, exposure to various environmental factors (radiation, chemicals, or certain viruses)

Question 3

Wide range of effects GENE MUTATION?

Answer 3

harmless, genetic disorders, development of diseases (cancer)

Question 4

substitution of 1 nucleotide, will not change the amino acid sequence

Answer 4

Silent

Question 5

change the amino acid sequence, but the replacement & the original amino acid have similar biochemical properties

Answer 5

Conservative

Question 6

replacement of amino acid w/ a biochemically different amino acid

Answer 6

Nonconservative

Question 7

terminates proteins prematurely when a nucleotide substitution produces a stop codon

Answer 7

Nonsense

Question 8

addition/deletion of nucleotides in a DNA sequence disrupts the reading frame

Answer 8

Frameshift

Question 9

purine replaces a purine or pyrimidine with a pyrimidine

Answer 9

Transition

Question 10

purine replaces a pyrimidine or pyrimidine with a purine

Answer 10

Transversion

Question 11

 Used to directly analyze the change in protein structure/function  Other uses:
 Metabolic defects where several genes are involved in the disease phenotype
 Detection of the actual protein/amino acid alterations

Answer 11

BIOCHEMICAL METHODS

Question 12

WHAT ARE THE 5 BIOCHEMICAL METHODS

Answer 12

1. Enzyme immunoassays 2. Immunohistochemistry 3. High-performance liquid chromatography (HPLC)
2. Gas chromatography
3. Mass spectrophotometry

Question 13

 Detects the presence of metabolites in the blood, urine, or other biological fluids

Answer 13

ENZYME IMMUNOASSAYS

Question 14

 Involve the use of specific antibodies or other ligands to detect the presence of the target molecules

Answer 14

ENZYME IMMUNOASSAYS

Question 15

Widely used EIA

Answer 15

enzyme-linked immunosorbent assays (ELISA)

Question 16

 Longstanding method that allows detection of protein abnormalities in situ

Answer 16

IMMUNOHISTOCHEMISTRY (IHC)

Question 17

Formalin-fixed paraffin-embedded tissue:

Answer 17

<5 micron slices (microtome)

Question 18

Fixation can affect tissue antigens altering/covering some epitopes  can be solved by antigen retrieval: enzyme digestion (proteinase K, chymotrypsin, pepsin, pronase) & heating tissue sections in water/buffer

Answer 18

Formalin-fixed paraffin-embedded tissue

Question 19

Snap frozen tissue (in isopentane, at -160ºC):

Answer 19

5 to 15-micron slices (cryostat inside of a chamber held at 20ºC)

Question 20

SNAP FROZEN TISSUE FIXATIVE

Answer 20

ACETONE

Question 21

Sections are dried & stored frozen

Answer 21

SNAP FROZ2N TISSUE

Question 22

Rehydration of the dried sections in of snap frozen tissue

Answer 22

phosphate buffered saline

Question 23

2 Imaging/microscopic observation of antibody binding requires a signal from the antibody:

Answer 23

Fluorescent signal
Colorimetric signal

Question 24

fluorescent molecules (fluorescein, Cy5, phycoerythrin)

Answer 24

Fluorescent signal

Question 25

substrate solution is added, oxidized by the enzymes (horseradish peroxidase/alkaline phosphatase)  most frequently used: red/ brown IHC staining

Answer 25

Colorimetric signal

Question 26

 Separation of molecules (NA & proteins) in solution through interaction with a solid support in the column

Answer 26

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

Question 27

2 PHASE OF HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

Answer 27

mobile phase (solvent) & stationary phase (solid support)

Question 28

increase resolution & lower separation time while using less solvent; faster flow rates (5 mL/min)

Answer 28

Ultra-HPLC (UHPLC)

Question 29

Separation of vaporized sample through a column of inert carrier gas (mobile phase) & liquid (stationary phase) that differently adsorbs molecules

Answer 29

GAS CHROMATOGRAPHY (GC)

Question 30

DETECTOR OF GAS CHROMATOGRAPHY (GC)

Answer 30

flame ionization detector

Question 31

 Used for detection of drugs & poisons & their metabolites in biological samples  May be coupled with MS to detect biomarkers of disease

Answer 31

GAS CHROMATOGRAPHY (GC)

Question 32

 Converts molecules to ions that can be moved in a magnetic field based on their charge & mass

Answer 32

MASS SPECTROMETRY (MS)

Question 33

2 ionization methods for large biomolecules (proteins) of MASS SPECTROMETRY (MS)

Answer 33

a. Electrospray ionization (ESI) b. Matrix-assisted laser desorption/ionization (MALDI)

Question 34

 High MW molecules  Ionized molecules are accelerated at a fixed point & allowed to drift through the flight tube to the detector

Answer 34

MALDI-TOF (time-of-flight) spectrometry

Question 35

 Combined w/ TOF offers flexibility in the ID & quantification of peptides

Answer 35

Surface-enhanced laser desorption/ionization (SELDI)

Question 36

 Performed on a variety of specimen types: blood/buccal cells  DNA mutations from single-base pair changes to large chromosomal rearrangements can be detected

Answer 36

NUCLEIC ACID ANALYSES

Question 37

simplified mutation detection (limiting specimens)

Answer 37

PCR amplification

Question 38

most definitive method for detecting mutations

Answer 38

DNA sequencing

Question 39

4 HYBRIDIZATION-BASED METHODS

Answer 39

Single-Strand Conformation Polymorphism (SSCP) SEQUENCING (POLYMERIZATION) BASED METHODS ENZYMATIC & CHEMICAL CLEAVAGE METHODS OTHER METHODS

Question 40

 Based on the preference of DNA to exist in a double-stranded state  Absence of the complementary strand: nucleic acids form intrastrand duplexes, 3D structure (conformer)  Shape: kinks, loops, bubbles, tail

Answer 40

Single-Strand Conformation Polymorphism (SSCP)

Question 41

 Different from the normal sequence (control) conformers  presence of gene mutation  Detected by silver stain, radioactivity, or fluorescence

Answer 41

Bands/peak patterns

Question 42

 Utilizes differences in the Tm of short sequences (20 bases) w/ 1 or 2 mismatches & those w/ no mismatches  Synthetic ss-probes (labeled) w/ normal/mutant target DNA sequence (immobilized) in a solution  At specific annealing temperatures & conditions (stringency) a. Probe will not bind to a near complementary target sequence w/ 1 or 2 mismatched bases b. Probe w/ perfect complementary sequence, will bind

Answer 42

Allele-Specific Oligomer Hybridization (ASO)

Question 43

 Method of analyzing the dissociation of dsDNA during the heating cycles  PCR amplicons in the presence of a DNA-specific fluorescent dye (EtBr, SYBR green, LC green) are heated (0.3ºC/sec)  Rise in the temperature, DNA duplexes begin to separate into single strands, losing the dye  Black line  targets w/ different mismatches to the hybridization probe

Answer 43

Melt Curve Analysis (MCA)

Question 44

overlaying peaks at expected Tm

Answer 44

Specimen w/ identical sequence

Question 45

2 or more peaks at different temperatures

Answer 45

Specimen w/ different sequence

Question 46

 High specificity  Uses fluorescent resonance energy transfer (FRET) probes  Raise in temperature, probes dissociate at specific Tm  donor no longer close to the acceptor  fluorescence drops

Answer 46

High-resolution melt-curve analysis (HRMCA)

Question 47

Tm lower than that of the probe & its perfect complement, or sequence difference between probe reference sequence & the test sequence

Answer 47

High-resolution melt-curve analysis (HRMCA) Indication of mutation

Question 48

Formed when single strands that are not completely complementary hybridize to 1 another

Answer 48

Heteroduplexes

Question 49

Can be resolved through polyacrylamide/agarose gel electrophoresis:

Answer 49

presence of bands different from a homozygous reference control is indicative of mutation

Question 50

Test DNA is fragmented by treatment with DNase before binding to the complementary probes on the array

Answer 50

High-density oligonucleotide arrays

Question 51

2 TYPES OF Hybridization formats

Answer 51

Standard tiling Redundant tiling

Question 52

base substitution is always in the 12th position from the 3’ end of the probe

Answer 52

Standard tiling

Question 53

same mutation is placed at different positions in the probe *Mutations are identified as indicated by which probes are bound

Answer 53

Redundant tiling

Question 54

uses sets of color-coded polystyrene beads in suspension as the solid matrix

Answer 54

Bead-array technology

Question 55

presence/absence of a mutation/polymorphism

Answer 55

Combination of bead color & test label

Question 56

 Detect point mutations & other SNPs  Primer 3’ end falls on the nucleotide to be analyzed  Must match the template perfectly to be extended by Taq polymerase  Presence/absence of the product = presence/absence of the mutation

Answer 56

Sequence-Specific (Primer) PCR (SSP-PCR)

Question 57

 Thermal cyclers w/ fluorescent detection  RT-PCR, using 2 probes labeled 3’ quencher molecules & different fluors on the 5’ ends (complementary to either normal/mutant sequence)  Presence of corresponding fluorescent signal indicates whether the test sequence is normal/mutant

Answer 57

Allelic Discrimination with Fluorogenic Probes

Question 58

 Can detect sequence alterations  Mutation changes the structure of a restriction enzyme target site/changes the size of a fragment  PCR-RFLP is used

Answer 58

Restriction Fragment Length Polymorphisms (RFLPs)

Question 59

 Heteroduplex analysis using duplex RNA  T7 or SP6 phage RNA polymerase  Detection of mutation: heteroduplexes form between normal & mutant transcripts  targets for cleavage by RNase enzymes (E. coli RNase & Aspergillus RNase T1)  Remaining dsRNA fragments can then be separated by agarose gel electrophoresis

Answer 59

Nonisotopic RNase Cleavage Assay (NIRCA)

Question 60

 Based on the characteristic enzymatic activity of cleavase  Premixed reagents (including cleavase) + standard 96 well-plate + test specimens + controls

Answer 60

Cleavage Assay

Question 61

recognizes the structure formed by hybridization of the normal/mutant probes to the test sequences

Answer 61

Cleavase

Question 62

Combination of methods to increase sensitivity & detection (RFLP with modified primers)

Answer 62

other metjods

Question 63

Array-based methods & massive parallel sequencing methods provide specific multiplex detection & sensitivity required for clinical applications

Answer 63

other methods